Abstract
Lactobacilli have been consistently associated with dental caries for decades; however, knowledge of this group of bacteria in the etiology of the disease is limited to quantitative elucidation. Nowadays, explicit identification of oral Lactobacillus species is possible, despite their taxonomic complexity. Here we describe a combined approach involving both cultivation and genetic methods to ascertain and characterize the diversity and abundance of the Lactobacillus population in the oral cavities of children with severe early childhood caries (S-ECC). Eighty 3- to 6-year-old children (40 S-ECC and 40 caries free) who were seeking dental care at the Pediatric Dental Clinic of Bellevue Hospital in New York City were invited to participate in this study. Clinical data on socio-demographic information and oral health behavior were obtained from the primary caregiver. The data included a detailed dental examination, children's medical history, and a questionnaire survey. Combined non-stimulated saliva and supra-gingival plaque samples were collected from each child and cultivated on selective media for quantitative measures of lactobacilli levels. The procedure for Lactobacillus species screening will include the random selection of 50 colonies per plate, extraction of DNA from each colony, and genotyping by arbitrarily primed polymerase chain reaction (AP-PCR). Each unique Lactobacillus AP-PCR genotype will be selected for taxonomic assessment by 16S rRNA gene sequencing analysis. Lactobacillus species will be identified by comparing the 16S rRNA sequences with the Ribosomal Database and the Human Oral Microbiome Database. Meanwhile, the same set of clinical samples will be independently subjected to genomic DNA isolation, 16S rRNA amplification with Lactobacillus genus-specific primers, sequencing, and taxonomic identification, both at genus and species levels with a customized pipeline. The distribution and phylogenetic differences of these Lactobacillus species will be compared between children with or without S-ECC. One of the main objectives of this study is to establish a study protocol for the identification and characterization of lactobacilli in the oral cavity. Future caries risk assessments can include lactobacilli counts (quantitative) and the presence/absence of specific cariogenic genetic signatures of a Lactobacillus species (qualitative) associated with S-ECC.
Highlights
Severe early childhood caries (S-ECC) is a aggressive form of dental caries associated with a history of maternal malnutrition and illness, tooth developmental defect [1] [2], excessive exposure to carbohydrates [3] and early infection by cariogenic microorganisms, predominantly mutans streptococci (MS) and lactobacilli [4]-[6]
Evidence based on decades of studies indicates that MS and lactobacilli are involved in the initiation of dental caries [7]
Recent studies have demonstrated that the microbiota of children with S-ECC differs significantly from that of their caries-free counterparts [11]-[13] and that lactobacilli comprise a significant portion of the cariogenic biota [5] [14] [15]
Summary
Severe early childhood caries (S-ECC) is a aggressive form of dental caries associated with a history of maternal malnutrition and illness, tooth developmental defect [1] [2], excessive exposure to carbohydrates [3] and early infection by cariogenic microorganisms, predominantly mutans streptococci (MS) and lactobacilli [4]-[6]. Studies have demonstrated that the presence of lactobacilli alone in caries lesions or saliva is insufficient to make inferences about their specific contribution in caries development. Other studies suggested that a number of Lactobacillus species can be niche-specific colonizers that vary according to the environment [21] [22] and that certain Lactobacillus species found in caries lesions show more cariogenic characteristics than others [20] [23]. It is plausible that a subgroup of caries-associated lactobacilli possesses niche-specific genetic elements linked to caries development, to S-ECC in particular. The genetic and adaptive characteristics of these Lactobacillus species are unclear, as well as the mechanisms used by the Lactobacillus species to assert their dominance within carious lesions
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